Introducing the human cell-free TX-TL system as a new prototyping platform for mammalian synthetic biology

A new in vitro workflow is presented for the characterisation of mammalian genetic regulatory elements in a transcription-translation (TX-TL) system. The reaction components including lysate of HeLa cells, DNA template, accessory proteins and energy mix, are assembled in wells of a 384 plate and continuously monitored for the expression of a fluorescent marker in a plate reader (Figure 1. B). Alternative setups include running the reaction on paper¹ or in microfluidic chip². In contrast, a usual transfection experiment in mammalian cells, to characterise a promoter for example, generally involves growing the cells, counting, plating, growing again, transfecting, waiting for the genes to be expressed, sample preparation and flow-cytometry, at least 4 days in total and many hours of work (Figure 1. A). Furthermore, TX-TL accepts linear DNA, as well as plasmid templates, which can replace the tedious steps of cloning by a simple PCR reaction for the preparation of DNA templates 3,4. The power of linear DNA combined with small-volume TXTL has been demonstrated in proteomic research where it is used to generate protein arrays⁵. For synthetic biology applications, the precise control over the components and the simplicity of the reaction (no cell division) provides an additional advantage for modelling⁶. Whereas prototyping in bacterial TX-TL is quickly gaining popularity in the synthetic biology community7 the analogous mammalian TX-TL method is yet to be established. The first aim of this study is to validate, for the first time to our knowledge, the approach of using the human cell-lysate based TX-TL to study various common types of gene regulation found in therapeutic gene circuits⁸: genome editing, transcription and translation regulation. Specifically, CRISPR/Cas9⁹ mediated DNA cleavage, an analogous method for dCas9 and Cpf1¹⁰ mediated repression of promoters¹⁰ and RBP mediated translational repression¹².